Bottom-Up Approach to Understand Chirality Transfer across Scales in Cellulose Assemblies

Cellulose is a polysaccharide that displays chirality across different scales, from the molecular to the supramolecular level. This feature has been exploited to generate chiral materials. To date, the mechanism of chirality transfer from the molecular level to higher-order assemblies has remained elusive, partially due to the heterogeneity of cellulose samples obtained via top-down approaches. Here, we present a bottom-up approach that uses well-defined cellulose oligomers as tools to understand the transfer of chirality from the single oligomer to supramolecular assemblies beyond the single cellulose crystal. Synthetic cellulose oligomers with defined sequences self-assembled into thin micrometer-sized platelets with controllable thicknesses. These platelets further assembled into bundles displaying intrinsic chiral features, directly correlated to the monosaccharide chirality. Altering the stereochemistry of the oligomer termini impacted the chirality of the self-assembled bundles and thus allowed for the manipulation of the cellulose assemblies at the molecular level. The molecular description of cellulose assemblies and their chirality will improve our ability to control and tune cellulose materials. The bottom-up approach could be expanded to other polysaccharides whose supramolecular chirality is less understood.


General Materials and Methods
All chemicals used were reagent grade and used as supplied unless otherwise noted. The automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. 1 Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in a staining solution (sugar stain: 10% H2SO4 in EtOH; CAM: 48 g/L ammonium molybdate, 60 g/L ceric ammonium molybdate in 6% H2SO4 aqueous solution). Flash column chromatography was carried out by using forced flow of the indicated solvent on Fluka Kieselgel 60 M (0.04 -0.063 mm). Analysis and purification was performed by reverse phase HPLC was performed by using an Agilent 1200 series. Products were lyophilized using a Christ Alpha 2-4 LD plus freeze dryer. 1 H, 13 C and HSQC NMR spectra were recorded on a Varian 400-MR (400 MHz), Varian 600-MR (600 MHz), or Bruker Biospin AVANCE700 (700 MHz) spectrometer. Spectra were recorded in CDCl3 by using the solvent residual peak chemical shift as the internal standard (CDCl3: 7.26 ppm 1 H, 77.0 ppm 13 C) or in D2O using the solvent as the internal standard in 1 H NMR (D2O: 4.79 ppm 1 H). 1 H NMR integrals of the resonances corresponding to residues at the reducing end are reported as non-integer numbers and the sum of the integrals of α and β anomers is set to 1. High resolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent) and a MALDI-TOF autoflex TM (Bruker). MALDI and ESI mass spectra were run on IonSpec Ultima instruments. IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotations were measured by using a Perkin-Elmer 241 and Unipol L1000 polarimeter.

Figure S1
BBs and solid supports used in this work.
Building block BB1a was purchased from GlycoUniverse (Germany). Building block BB1b was synthesized according to previously reported procedures. 2 Merrifield resin equipped with a photocleavable linker (L1, loading 0.35 mmol/g) was prepared according to previous literature. 3 The synthesis of BB2 is reported herein (Scheme S1).

Synthesis of S-2
S-1 was synthesized according to previously reported procedures. 4 S-1 (1.06 g, 3.40 mmol) was dissolved in MeOH (40 mL), di-n-butyltin oxide (Bu2SnO) (1.02 g, 4.10 mmol) was added and the reaction mixture (white suspension) heated at reflux (65 °C) under vigorous stirring for 22 h. The reaction mixture (clear solution) was then cooled, concentrated under reduced pressure and the crude product was used in the next step without further purification. The crude was dissolved in DMF (20 mL). Benzyl bromide (0.49 mL, 4.13 mmol) and cesium (I) fluoride (CsF) (670 g, 4.41 mmol) were added and the clear solution stirred at RT for 24 h under Ar atmosphere. The cloudy reaction mixture was diluted with EtOAc and the organic layer was passed through a short plug of silica gel and concentrated under reduced pressure. The crude product was diluted with EtOAc and the organic layer washed once with an aqueous solution of KF (1 M), once with water, once with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography (Hexane : Acetone = 3:12:11:1) to yield S-2 as a white solid (0.78 g, 57%).

Synthesis of S-3
S-2 (775 mg, 1.93 mmol) was dissolved in anhydrous DCM (40 mL) under Ar atmosphere. Triethylamine (NEt3) (2.86 mL, 0.58 mmol) and 4-dimethylaminopyridine (DMAP) (70 mg, 0.58 mmol) were added to the solution, while stirring. Benzoyl chloride (BzCl) (350 μL, 3.03 mmol) was slowly added at 0°C and the reaction allowed to RT. After 18 h the reaction was diluted with DCM and quenched with a saturated aqueous solution of NaHCO3. The organic layer was washed three times with a saturated aqueous solution of NaHCO3 and once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified through a short plug of silica (EtOAc isocratic) and recrystallized from Hexane : EtOAc to yield S-3 as a white solid (708 mg, 72%).

General materials and methods
The automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. 1 All solvents used were HPLC-grade. The solvents used for the building block, activator, TMSOTf and capping solutions were taken from an anhydrous solvent system (J.C. Meyer) for moisture-sensitive solutions. The building blocks were co-evaporated three times with toluene and dried for 1 h under high vacuum before use. Oven-heated, argon-flushed flasks were used to prepare all moisture-sensitive solutions. Activator, capping, deprotection, acidic wash and building block solutions were freshly prepared and kept under argon during the automation run. All yields of products obtained by AGA were calculated on the basis of resin loading. Resin loading was determined following previously established procedures. 5

Modules for automated synthesis Module A: Resin Preparation for Synthesis (20 min)
All automated syntheses were performed on 0.0125 mmol scale. Resin (L1, 36 mg or L2, 34 mg) was placed in the reaction vessel and swollen in DCM for 20 min at room temperature prior to synthesis. During this time, all reagent lines needed for the synthesis were washed and primed. After the swelling, the resin was washed with DMF, THF, and DCM (three times each with 2 mL for 25 s).

Module B: Acidic Wash with TMSOTf Solution (20 min)
The resin was swollen in 2 mL DCM and the temperature of the reaction vessel was adjusted to -20 °C. Upon reaching the low temperature, TMSOTf solution (1 mL) was added drop wise to the reaction vessel. After bubbling for 3 min, the acidic solution was drained and the resin was washed with 2 mL DCM for 25 s. The building block solution (0.06 mmol of BB in 1 mL of DCM per glycosylation) was delivered to the reaction vessel. After the set temperature was reached, the reaction was started by drop wise addition of the TMSOTf solution (1.0 mL, stoichiometric). After completion of the reaction, the solution was drained and the resin washed with DCM (six times, each with 2 mL for 25 s). The temperature of the reaction vessel was increased to 25 °C for the next module. The resin was washed with DMF (two times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. 2 mL of Pyridine solution (10% in DMF) was delivered into the reaction vessel. After 1 min, the reaction solution was drained and the resin washed with DCM (three times with 3 mL for 25 s). 4 mL of capping solution was delivered into the reaction vessel. After 20 min, the reaction solution was drained and the resin washed with DCM (three times with 3 mL for 25 s).

Module E: Fmoc Deprotection (9 min)
The resin was washed with DMF (three times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. 2 mL of Fmoc deprotection solution was delivered to the reaction vessel and kept under Ar bubbling. After 5 min, the reaction solution was drained and the resin washed with DMF (three times with 3 mL for 25 s) and DCM (five times each with 2 mL for 25 s). The temperature of the reaction vessel was decreased to -20 °C for the next module.

Post-AGA manipulations Module F: On-resin Methanolysis
The resin was suspended THF (4 mL). MeONa in MeOH (0.5 M, 0.4 mL) was added and the suspension was gently shaken at room temperature. After micro-cleavage (see Module G1) indicated the complete removal of benzoyl groups, the resin was repeatedly washed with MeOH (2mL x 3) and DCM (2mL x 3). For D7, D8 and D9 a higher amount of MeONa in MeOH (0.5 M, 0.8 mL) was used.

Module G: Cleavage from Solid Support
The oligosaccharides were cleaved from the solid support using a continuous-flow photoreactor as described previously. 8

Module G1: Micro-cleavage from Solid Support
Trace amount of resin (around 20 beads) was dispersed in DCM (0.1 mL) and irradiated with a UV lamp (6 W, 356 nm) for 10 min. ACN (10 µL) was then added to the resin and the resulting solution analyzed by MALDI.

Module H1: Hydrogenolysis
The crude compound obtained from Module G was dissolved in 2 mL of EtOAc:tBuOH:H2O (2:1:1). 100% by weight Pd/C (10%) was added and the reaction was stirred in a pressurized reactor under 4 bar pressure of H2.
The reaction progress was monitored to avoid undesired side products formation (i.e. degradation of reducing end). 9 Upon completion, the reaction was filtered and washed with EtOAc, tBuOH and H2O (4 mL each). The filtrates were concentrated in vacuo. For D8 and D9, the products were kept in solution to prevent aggregation. The organic phase was removed from the reaction mixture and the aqueous phase was washed with EtOAc two times. The remaining aqueous solution was concentrated to about half the volume to remove traces of EtOAc and tBuOH.

Module H2: Hydrogenolysis at ambient pressure
The crude compound obtained from Module G was dissolved in 2 mL of EtOAc:tBuOH:H2O (2:1:1). 100% by weight Pd/C (10%) was added and the reaction was stirred in a flask equipped with a H2 balloon. The reaction progress was monitored to avoid undesired side products formation. Upon completion, the reaction was filtered and washed with EtOAc, tBuOH, ACN and H2O (4 mL each). The filtrates were concentrated in vacuo.

Module I: Purification
The purification of the crudes was conducted using a C18 silica column or reverse phase HPLC (Agilent 1200 Series, Method B and Method C). The pure compound was analyzed using analytical HPLC (Agilent 1200 Series, Method A). Following final purification, all deprotected products were lyophilized on a Christ Alpha 2-4 LD plus freeze dryer prior to characterization.

Figure S2
Collection of cellulose analogues synthesized by AGA.

D5
Step Modules Notes

D6
Step Modules Notes Analytical data for D6 were in good agreement with previously reported data.

D7
Step Modules Notes Analytical data for D7:

D8
Step Modules Notes

L3D3
Step Modules Notes

L2D4
Step Modules Notes

LD6L
Step Modules Notes

LD5L
Step Modules Notes

LD6
Step Modules Notes

Oligosaccharides self-assembly 4.1 Solubility measurement
The lyophilized powder was weighed, MilliQ water was added in portions, and the mixture was bubbled with N2 for 30 s. Upon visual disappearance of the precipitate a range of solubility was calculated. Water addition was stopped when the calculated solubility was < 0.5 mg/mL.

Compound
Mass (

TEM imaging
Transmission electron microscopy was performed using a JEM 2100Plus transmission electron microscope (Jeol, Japan) operated at an accelerating voltage of 200 kV or a FEI Talos L120C (Thermo Fisher, USA) operated at an accelerating voltage of 120 kV (LaB6 cathode). Drops (3-4 μL) of aqueous suspensions (or MeOH suspensions) of crystallites were deposited on glow-discharged carbon-coated copper grids. Negative staining (2% uranyl acetate aqueous solution) was applied (3 μL drop) to the grid after the sample was deposited, allowed to settle for approximately 1 min, and then carefully blotted away with filter paper. Negative staining of the TEM grids has been used to enhance contrast only where specified in the images reported below. 10

Figure S5
Representative TEM images of D6 obtained from aqueous suspension (1 mg/mL) using negative staining.

Figure S6
Representative TEM images of D6 obtained from aqueous suspension (1 mg/mL). The red arrows indicate the assemblies where the features indicating a twisted morphology are more evident.

Figure S7
Representative TEM images of D6 twisted assemblies at high magnifications obtained from aqueous suspension (1 mg/mL).

Figure S8
Excerpts of TEM images of D6 twisted assemblies obtained from aqueous suspension. Scale bar 500 nm.

Figure S9
Representative TEM images of L6 obtained from aqueous suspension (1 mg/mL) using negative staining.

Figure S10
Representative TEM images of L6 obtained from aqueous suspension (1 mg/mL). The red arrows indicate the assemblies where the features indicating a twisted morphology are more evident.

Figure S11
Representative TEM images of L6 twisted assemblies at high magnifications obtained from aqueous suspension (1 mg/mL).

Figure S12
Excerpts of TEM images of L6 twisted assemblies obtained from aqueous suspension. Scale bar 500 nm.

Figure S13
A-B) Tem image and electron diffraction analysis of L6 bundles obtained from aqueous suspension (1 mg/mL). The pattern was assigned to the cellulose II allomorph. Scale bar 500 nm.

Figure S14
A) Representative TEM images of L6 obtained from aqueous suspension (1 mg/mL) showing a fan-like arrangement of the stacking platelets (red arrow). B) The fan-like arrangement of the stacking platelets was interpreted as a rotation between the (001) planes.

Figure S15
Representative TEM images of D7 obtained from aqueous suspension (1 mg/mL) using negative staining.

Figure S16
Representative TEM images of D7 obtained from aqueous suspension (1 mg/mL).

Figure S17
Representative TEM images of D8 obtained from aqueous suspension (1 mg/mL) using negative staining.

Figure S18
Representative TEM images of D8 obtained from aqueous suspension (1 mg/mL).

Figure S20
Representative TEM images of L6 obtained from aqueous suspension (1 mg/mL or 0.5 mg/mL) varying the evaporation rate. A) The grid was allowed to dry (< 1 h). B) The solvent evaporation rate was slowed down by covering the grid with a petri dish (ca. 3 h).

Figure S21
Representative TEM images of L6 (A-C) and D6 (D) obtained from aqueous suspension at different concentration. The solvent evaporation rate was slowed down by covering the grid with a petri dish (ca. 3 h).

Figure S24
Representative TEM images of LD6 platelets obtained from MeOH suspension after recrystallization.

Figure S25
Representative TEM images of LD6 bundles obtained from aqueous suspension (1 mg/mL).

Figure S27
A, B) Electron diffraction analysis of LD6L obtained from aqueous suspension (1 mg/mL). The pattern was assigned to the cellulose IVII allomorph. C, D) Electron diffraction analysis of LD6L obtained from aqueous suspension after recrystallization. The pattern was assigned to the cellulose II allomorph.

Figure S28
Representative TEM images of LD6L platelets obtained from MeOH suspension after recrystallization.

Figure S29
Representative TEM images of LD6L bundles obtained from MeOH suspension after recrystallization.

Figure S30
Representative TEM images of LD5L obtained from aqueous suspension (1 mg/mL) using negative staining.

Figure S31
Representative TEM images of LD5L obtained from MeOH suspension after recrystallization.

AFM and SEM imaging
Atomic force microscopy was performed with a JPK NanoWizard 4 AFM in tapping mode (AC mode) or a Dimension ICON instrument (Bruker) in pulse force (PeakForce) mode using SNL-10 A tip (0.35 N/m, 65 kHz, Bruker) or Arrow NCR tip (42 N/m, 285 kHz, Nano World). The samples were prepared as follows: approximately 0.1 mg of the lyophilized powder was weighed (in a glass or plastic vial), diluted with MilliQ water to reach the concentration of 1 mg/mL. For AFM imaging, the solution was further diluted with MilliQ water to reach the concentration of 0.1 mg/mL. Drops of aqueous suspensions were deposited on freshly cleaved mica or on glow-discharged (0.8 mbar, 30 mA for 20 s using air) silicon wafer and dried at room temperature. AFM images were collected with 1024 x 1024 pixels/frame and analyzed with the JPK Data Processing software.
Scanning electron microscopy (SEM) images were obtained with a Gemini SEM, LEO 1550 system with cold field emission gun operation at 3 kV. All the samples were coated with Au/Pd. The hydrophilic glass substrate was prepared by treating a round shape glass slide with an HCl aqueous solution (0.5 M) for 1 h and then rinsed with water.

Figure S32
Representative AFM images of D6 obtained from aqueous suspension (0.1 mg/mL) drop casted on freshly cleaved mica.

Figure S33
Representative AFM images of D7 obtained from aqueous suspension (0.1 mg/mL) drop casted on freshly cleaved mica.

Figure S34
Representative AFM images of D8 obtained from aqueous suspension (approx. 0.1 mg/mL) drop casted on freshly cleaved mica (A-C) and silicon wafer (D-F).

Figure S35
Representative AFM images of L6 obtained from aqueous suspension (0.1 mg/mL) drop casted on freshly cleaved mica.

Figure S36
AFM height histograms for single-layer platelets. The height of the platlets was measured manually to avoid artifacts caused by edges or overlays.

Figure S37
A) Representative TEM image obtained from D6 aqueous suspension (1 mg/mL). B-C) AFM height image obtained from the same TEM grid.